JP6608392B2 - Genetically modified microorganisms for improved production of fine chemicals in sucrose - Google Patents

Description

  The present invention relates to modified microorganisms, methods for producing organic compounds and the use of modified microorganisms.

  Organic compounds such as small dicarboxylic acids having 6 or fewer carbons are commercially important chemicals with many uses. For example, small divalent acids include 1,4-2 valent acids such as succinic acid, malic acid and tartaric acid, and the 5-carbon itaconic acid. Other divalent acids include 2-carbon oxalic acid, 3-carbon malonic acid, 5-carbon glutaric acid and 6-carbon adipic acid, and many derivatives of such divalent acids are present as well.

  As a group, small diacids have some chemical similarity and their use in polymer production can provide specialized characteristics for the resin. Such versatility allows them to easily fit into the downstream chemical infrastructure market. For example, 1,4-2 valent acid molecules include various industrial chemicals (tetrahydrofuran, butyrolactone, 1,4-butanediol, 2-pyrrolidone) and succinate derivatives succinamide, succinonitrile, diaminobutane. And satisfies many of the large-scale uses of chemical maleic anhydride in that it is converted to an ester of succinate. Tartaric acid has many uses in the food, leather, metal and printing industries. Itaconic acid forms the starting material for the production of 3-methylpyrrolidone, methyl-BDO, methyl-THF and the like.

  In particular, succinic acid or succinate—these terms are used interchangeably herein—because they have been used as precursors to many industrially important chemicals in the food, chemical and pharmaceutical industries. Has attracted interest. In fact, a US Department of Energy report reports that succinic acid is one of the top 12 chemical building blocks produced from biomass. Therefore, the ability to make divalent acids in bacteria has important commercial significance.

  WO-A-2009 / 024294 is a succinic acid producing bacterial strain that is originally isolated from rumen and is a member of the family Pasteurellaceae having the ability to utilize glycerol as a carbon source, and to maintain said ability With the deposit number of the derived mutant, especially DSM 18541 (ID 06-614), deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) and named DD1 Also disclosed are bacterial strains that have the ability to produce succinic acid. The DD1 strain belongs to the Basfia succiniciproducens species and the family Pasteurellaceae classified by Kuhnert et al., 2010. Mutations of these strains in which the ldhA gene and / or pflD or pflA gene is disrupted are disclosed in WO-A-2010 / 092155, these variants are the DD1 disclosed in WO-A-2009 / 024294 Compared to the wild type, it is characterized by a significantly increased production of succinic acid from carbon sources such as glycerol or a mixture of carbohydrates such as glycerol and maltose under anaerobic conditions.

  However, bio-based succinates still face the challenge of becoming cost competitive with petrochemical-based alternatives. In order to develop bio-based industrial production of succinic acid, it is possible to grow the cells in a low-cost medium and use a low-cost, cost-effective range of working strains, so that the cheapest available raw materials can be used. It would be important to metabolize the sugar feedstock to produce succinic acid in good yield.

  Sucrose (commonly known as sugar) is a disaccharide composed of glucose and fructose, is a very rich carbon source in nature, and is produced from all plants that have photosynthetic ability. In particular, sugar cane and sugar beet contain large amounts of sucrose, and over 60% of the world's sucrose is currently produced from sugar cane. In particular, sucrose is produced at a very low cost because it is produced industrially through a simple process of evaporation / concentration of the extract obtained by mechanical compression of sugarcane. Thus, sucrose as a raw material for producing chemical compounds via microbial fermentation is inexpensive and functions to protect cell membranes from the external environment containing large amounts of the desired metabolite, and thus Kilimann et al. Biochimica et Biophysica Acta, 1764, 2006) produces high concentrations of the desired metabolite.

  Although sucrose is an excellent raw material having the above advantages including low price and the function of protecting microorganisms from the external environment, the disadvantage of this carbon source is that many microorganisms It can be seen in the fact that it does not have a complete mechanism for transporting to cells, degrading the transported sucrose and linking the degradation product to the thawing system, and therefore sucrose cannot be used as a carbon source. Even in the case of microorganisms having a mechanism for using sucrose, the rate of incorporation and decomposition of sucrose as a carbon source is very low, so that a desired metabolite cannot be produced efficiently.

International Publication No. 2009/024294 International Publication No. 2010/092155

Kilimann et al. (Biochimica et Biophysica Acta, 1764, 2006)

  The object of the present invention was therefore to eliminate the disadvantages of the prior art.

  In particular, the object of the present invention is to provide a microorganism that can be used for the fermentative production of organic compounds such as succinic acid and can efficiently utilize sucrose as a main carbon source without sacrificing growth rate or yield. Was to do. Preferably, the microorganism can use many low-cost carbon sources and can produce an organic compound such as succinic acid in an excellent yield. Compared to prior art recombinant microorganisms used for fermentative production of succinic acid, the microorganisms of the present invention have increased succinic acid yield and increased carbon during cell growth on sucrose as the main carbon source. Should be characterized by yield.

The contribution to achieve the above objective is compared to the wild type compared to the wild type,
-Reduced activity of the enzyme encoded by the ptsA gene,
A modified microorganism having a reduced activity of the enzyme encoded by the ptsH gene, or a reduced activity of the enzyme encoded by the ptsA gene and a reduced activity of the enzyme encoded by the ptsH gene, The wild type from which the microorganism is derived is provided by a modified microorganism belonging to the family Pasteurellaceae.

The contribution to achieve the above objectives is in particular:
ptsA gene or a part thereof,
ptsH gene or part thereof, or
Modifications in which the ptsA gene or a part thereof and the ptsH gene or a part thereof are deleted, or the regulatory elements of those genes or at least a part thereof are deleted, or in which at least one cooking is introduced into those genes A wild-type microorganism from which the modified microorganism is derived is provided by a modified microorganism belonging to the family Pasteurellaceae.

  Surprisingly, the ptsA gene (this enzyme depends on phosphoenolpyruvate) in microorganisms belonging to the family Pasteurellae, for example by deletion of the ptsA gene or parts thereof and / or deletion of the ptsH gene or parts thereof Reduced by the activity of the enzyme encoded by the energy-coupled phosphotransferase system) and / or by the ptsH gene, which is the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system Reduction of the activity of the enzyme, especially when the modified microorganism is grown on sucrose as a carbon source, compared to this enzyme or a corresponding microorganism in which the activity of these enzymes has not decreased, organics such as succinic acid Result in modified microorganisms characterized by increased yields of compounds It was found. This is because the phosphoenolpyruvate-dependent phosphotransferase system (ie, the PTS system) is fructose in microorganisms belonging to the family Pasteurellae, for example the family of the genus Basfia, in particular the family of the species Basfia succiniciproducens. It is actually surprising because it is involved in cellular uptake. When a basophil strain is cultured in sucrose, this disaccharide is hydrolyzed intracellularly to yield glucose-6-phosphate (fatphas) and fructose. However, fructose is secreted after hydrolysis and is taken up again into the cell using the fructose PTS system. Thus, those skilled in the art will know that inactivation of the ptsA gene and / or ptsH gene can transport at least a portion of this disaccharide (ie, fructose) to the cell when the cell is cultured in sucrose as the main carbon source. This would be thought to result in a reduced formation of succinic acid.

FIG. 1 shows a schematic map of plasmid pSacB (SEQ ID NO: 17). FIG. 2 shows a schematic map of plasmid pSacBΔldhA (SEQ ID NO: 18). FIG. 3 shows a schematic map of plasmid pSacBΔpflA (SEQ ID NO: 19). FIG. 4 shows a schematic map of plasmid pSacB pykA1 (SEQ ID NO: 20). FIG. 5 shows a schematic map of plasmid pSacB wcaJ ^* (SEQ ID NO: 21). FIG. 6 shows a schematic map of plasmid pSacBΔptsA (SEQ ID NO: 22). FIG. 7 shows a schematic map of plasmid pSacBΔptsH (SEQ ID NO: 23).

  In the context of the expression “modified microorganism having a reduced activity of the enzyme encoded by the x gene compared to (its) wild type”, where the x gene is the ptsA or ptsH gene and optionally As described, the term “wild type” (which is the ldhA gene, the pflA gene, the pflD gene, the wcaJ gene and / or the pykA gene) refers to a microorganism in which the activity of the enzyme encoded by the x gene is not reduced, ie its genome. Refers to a microorganism that exists in the same state as before the introduction of the genetic modification of the x gene. Preferably, the expression “wild type” refers to a microorganism (eg, bacterium, yeast cell, fungal cell, etc.) in which its genome, in particular its x gene, is present in a naturally occurring state as a result of evolution. The term is used for both whole microorganisms and individual genes. As a result, the term “wild type” preferably does not include microorganisms, or genes, in particular whose gene sequence has been at least partially artificially modified by recombinant methods. Thus, the term “modified microorganism” refers to an altered, modified or different genotype and / or phenotype as compared to the natural wild-type microorganism from which it is derived (eg, genetic modification is a microorganism). Of the encoding nucleic acid sequence), including genetically altered, modified or engineered (eg, genetically engineered) microorganisms. According to certain preferred embodiments of the modified microorganism according to the invention, the modified microorganism is a recombinant microorganism, which means that the microorganism has been obtained using recombinant DNA. The expression “recombinant DNA” as used herein is a laboratory method (molecular cloning) that collects genetic material from multiple sources and creates sequences that would otherwise not be found in an organism. Refers to the DNA sequence resulting from the use of An example of such recombinant DNA is a plasmid into which a heterologous DNA sequence has been inserted.

  The wild type from which the microorganisms according to the invention are derived belongs to the family Pasteurellaceae. Pasteurellaceae include a large family of gram-negative proteobacteria with members ranging from bacteria such as Haemophilus influenzae to symbiotic organisms of animals and humans. Most members live as commensals on the mucosal surfaces of birds and mammals, especially in the upper respiratory tract. Pasteurellaceae are typically rod-shaped and are a well-known group of facultative anaerobes. They can be distinguished from the related Enterobacteriaceae family by the presence of oxidases, and can be distinguished from most other similar bacteria by the absence of flagella. Pasteurellaceae bacteria are classified into a number of genera based on metabolic characteristics and 16S and 23S RNA sequences. Many of the Pasteurellaceae contain the pyruvate formate lyase gene and have the ability to anaerobically ferment carbon sources to organic acids.

  According to a preferred specific embodiment of the modified microorganism according to the invention, the wild type from which the modified microorganism is derived belongs to the genus Basfia, and the wild type from which the modified microorganism is derived is represented by Basfia succinici produce (Basfia). It is particularly preferred to belong to the succiniciproducens) species.

  Most preferably, the wild type from which the modified microorganism according to the present invention is derived is DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) (Germany) August 2006 under the Budapest Treaty. Bastia succinici producers DD1 strain with deposit number DSM 18541 deposited on the 11th. This strain was originally isolated from bovine rumen of German origin. Pasteurellaceae bacteria can be isolated from the gastrointestinal tract of animals and preferably mammals. Bacterial strain DD1 can in particular be isolated from bovine rumen and can utilize glycerol (including unpurified glycerol) as a carbon source. Further strains of the genus Basfia that can be used to prepare modified microorganisms according to the present invention include the Basfia strain commercially available from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH as DSM number 22022, or the University of Gothenburg Culture Collection (CCUG, University of Deposited on February 27, 2009 in Gothenburg, Department of Clinical Bacteriology, Guldhedsgatan 10, SE-413 46 (Sweden), Sweden, with deposit numbers CCUG 57335, CCUG 57762, CCUG 57763, CCUG 57764, CCUG 57765 or CCUG 57766 It is a bus sphere stock. The strain was originally isolated from bovine rumen of German or Swedish origin.

  In this context, the wild-type from which the modified microorganism according to the invention is derived exhibits a sequence homology of at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9% with SEQ ID NO: 1 or SEQ ID NO: 1 It is particularly preferred to have 16S rDNA of The wild-type from which the modified microorganism according to the invention is derived comprises SEQ ID NO: 2 or 23S rDNA of the sequence showing at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9% sequence homology with SEQ ID NO: 2. It is also preferable to have it.

  The identity in the percent values referred to for the various polypeptides or polynucleotides used for the modified microorganisms according to the invention is preferably calculated as the identity of the residues over the entire length of the aligned sequence, for example the default parameters, Gap Open (Gap Opening Penalty): 10.0,-Gap Extend (Gain Extension Penalty): 0.5, Data File (scoring the matrix file included in the package): Bioinformatics Software Package EMBOSS (Version 5.0) using EDNAFUL .0, http: //emboss.sourceforgenet/what/) using calculated needles (for fairly similar sequences).

  It should be noted that the modified microorganisms according to the present invention can be derived not only from the wild-type microorganisms described above, but particularly from the Basfia succinici produce DD1 strain, as well as variants of these strains. In this context, the expression “mutant strain” includes all strains having the same or essentially the same characteristics as the wild type strain. In this context, the mutant 16S rDNA is at least 90%, preferably at least 95%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.6% of the wild type from which the mutant is derived. It is particularly preferred to have an identity of at least 99.7%, more preferably at least 99.8% and most preferably at least 99.9%. The 23S rDNA of the mutant is at least 90%, preferably at least 95%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably the wild type from which the mutant is derived. It is also particularly preferred to have at least 99.7% identity, more preferably at least 99.8% and most preferably at least 99.9%. Mutants in the sense of this definition can be obtained, for example, by treating wild-type strains with mutagens, X-rays or UV light.

  The modified microorganism according to the present invention has a reduced activity of the enzyme encoded by the ptsA gene, a reduced activity of the enzyme encoded by the ptsH gene, or a reduction of the enzyme encoded by the ptsA gene compared to the wild type. And a reduced activity of the enzyme encoded by the ptsH gene.

Reduction of the enzyme activity (delta _activity) is defined by the following:
Here, when the Δ _activity is determined, the wild type activity and the activity of the modified microorganism are determined under exactly the same conditions. For example, Reizer et al: “Evidence for the presence of heat-stable protein (HPr) and ATP-dependent HPr kinase in heterofermentative lactobacilli lacking phosphoenolpyruvate: glycose Proc. Nadl. Acad. Sci. USA; Vol. 85, pages 2041-2045 (1988) ".

  Reduced activity of enzymes disclosed herein, particularly reduced activity of enzymes encoded by the ptsA gene and / or ptsH gene, ldhA gene, pflA gene, pflD gene and / or wcaJ gene, Reduced enzyme activity by at least 50%, or at least 90% enzyme activity, or more preferably at least 95% enzyme activity reduction, or more preferably at least 98% compared to the activity of said enzyme in the wild type of A reduction in enzyme activity, or even more preferably at least 99% reduction in enzyme activity, or even more preferably at least 99.9% reduction in enzyme activity. In the case of the pykA gene, the reduced activity is preferably a 0.1-99% reduction in enzyme activity, or at least 15%, or at least 25%, or at least compared to the activity of said enzyme in the wild type of the microorganism. 35%, or at least 45%, or at least 55%, or at least 65%, or at least 75% or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90 %, Or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of the enzyme activity It is a reduction. Preferably, the reduction in the activity of the enzyme encoded by the pykA gene is in the range 15-99%, more preferably in the range 50-95%, even more preferably in the range 90-99%. The term “reduced activity of the enzyme encoded by the x gene” also encompasses modified microorganisms that do not have detectable activity of this particular enzyme.

  The term “reduced activity of an enzyme” refers to a lower level of enzyme expressed by the wild type of the microorganism, eg, by the genetically manipulated (eg, genetically engineered) microorganism. Expression. Genetic treatments to reduce the expression of the enzyme include, but are not limited to, a deletion of a gene encoding the enzyme or a portion thereof, eg, by removal of a strong promoter or repressed promoter. Alteration or modification of regulatory sequences or sites involved in the expression of genes to be altered, transcription of genes encoding enzymes and / or alteration of proteins involved in translation of gene products (eg regulatory proteins, suppressors, enhancers and transcriptional activators), Or any other conventional means of reducing the expression of a particular gene that is routine in the art (including but not limited to the use of antisense nucleic acid molecules or other methods that knock out or block expression of the target protein Means). In addition, destabilizing elements can be introduced into the mRNA, or genetic modifications can be introduced that result in deterioration of the ribosome binding site (RBS) of the RNA. It is also possible to change the codon usage of the gene so that translation efficiency and speed are reduced.

  Reduced activity of the enzyme can also be achieved by introducing one or more genetic mutations that result in reduced activity of the enzyme. Furthermore, reducing the activity of an enzyme can also include inactivation (or reduced expression) of an activating enzyme that is essential to activate the enzyme whose activity is reduced. By the latter approach, enzymes whose activity is reduced are preferably kept in an inactivated state.

  Microorganisms with reduced activity of the enzyme encoded by the ptsA gene and / or the ptsH gene can occur naturally, i.e. by spontaneous mutation. The microorganism can be modified to lack or have a significantly reduced activity of the enzyme encoded by the ptsA gene and / or the ptsH gene by various techniques such as chemical treatment or radiation. For this purpose, the microorganism will be treated, for example, with mutagenic chemicals, X-rays or UV light. In a later step, microorganisms with reduced activity of the enzyme encoded by the ptsA gene and / or the ptsH gene will be selected. A modified microorganism mutates, disrupts or excises the ptsA gene and / or the ptsH gene in the genome of the microorganism, or an enzyme with reduced activity compared to the enzyme encoded by the wild-type gene. It can also be obtained by homologous recombination techniques aimed at replacing the corresponding corresponding gene.

  According to a preferred embodiment of the modified microorganism according to the invention, the reduction of the activity of the enzyme encoded by the ptsA gene and / or the ptsH gene is achieved respectively by modification of the ptsA gene and / or the ptsH gene, wherein this genetic modification Is preferably a deletion of a ptsA gene and / or ptsH gene or at least a part of these genes, a ptsA gene and / or a ptsH gene regulatory element such as a promoter sequence, or a deletion of a part of these regulatory elements, or This is achieved by introducing at least one mutation into the ptsA gene and / or the ptsH gene.

  The following describes suitable techniques for recombination, particularly for introducing mutations or deleting sequences.

  This technique is sometimes referred to herein as “Campbell recombination” (Leenhouts et al., 1989, Appl Env Microbiol 55, 394-400). As used herein, “Campbell in” refers to the entire circular double-stranded DNA molecule (eg, a plasmid) being integrated into a chromosome by a single homologous recombination event (cross-in event), thereby , Refers to a transformant of the original host cell in which the linear form of the circular DNA molecule is efficiently inserted into a first DNA sequence on a chromosome homologous to the first DNA sequence of the circular DNA molecule. “Campbelled in” refers to a linearized DNA sequence that is integrated into the chromosome of a “Campbell in” transformant. “Campbell in” contains a duplication of the first homologous DNA sequence, each copy containing and surrounding a copy of the homologous recombination crossover point.

  As used herein, “Campbell out” refers to a second DNA sequence in which a second homologous recombination event (cross-out event) is contained on the linearized insert DNA of “Campbell in” DNA; Refers to a cell that is a descendant of a “Campbell in” transformant occurring between a second DNA sequence derived from a chromosome homologous to the second DNA sequence of the linearized insert. Here, the second recombination event results in the deletion (release) of a portion of the incorporated DNA sequence, but importantly, a portion of the incorporated “Campbell in” DNA (which is only a single unit). Can also be left in the chromosome, compared to the original host cell, the “Campbell out” cell contains one or more intentional changes in the chromosome (eg, single base substitution, multiple bases) Substitution, insertion of a heterologous gene or DNA sequence, insertion of a further copy of a homologous or modified homologous gene, or insertion of a DNA sequence comprising two or more of these examples listed above). “Campbell out” cells preferably grow in the presence of a gene, eg, about 5% to 10% sucrose, contained in a portion of the “Campbell in” DNA sequence (the portion that is desired to be released). It is obtained by counter-selection against the Bacillus subtilis sacB gene, which is lethal when expressed in cells. With or without counterselection, the desired “Campbell out” cells can be of any screenable phenotype, including but not limited to colony morphology, colony color, antibiotic resistance The presence or absence of DNA, presence or absence of certain DNA sequences by polymerase chain reaction, presence or absence of auxotrophy, presence or absence of enzymes, colony nucleic acid hybridization, antibody screening, etc. Or can be identified. The terms “Campbell in” and “Campbell out” can also be used as various tense verbs to refer to the method or process described above.

  It is understood that homologous recombination events leading to “Campbell in” or “Campbell out” can occur over a range of DNA bases within a homologous DNA sequence, and the homologous sequences are identical to each other at least in part of this range. Thus, it is usually not possible to identify the exact location where the crossover event occurred. That is, it cannot be accurately specified which sequence is derived from the inserted DNA and which sequence is derived from the chromosomal DNA. In addition, the first homologous DNA sequence and the second homologous DNA sequence are usually separated by a partially non-homologous region, and it is this non-homologous region that remains on the chromosome of the “Campbell out” cell.

  Preferably, the first and second homologous DNA sequences are at least about 200 base pairs long and can be up to several thousand base pairs long. However, the procedure can be made to work with shorter or longer sequences. For example, the length of the first and second homologous sequences can be about 500-2000 bases, and obtaining “Campbell out” from “Campbell in” makes the first and second homologous sequences approximately the same length. Preferably by less than 200 base pairs, most preferably by making the shorter of the two base pairs at least 70% of the longer length.

The ptsA gene whose activity is reduced in the modified microorganism according to the present invention is preferably:
a1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 3;
b1) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 4;
c1) at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% with the nucleic acid of a1) or b1) A nucleic acid that is at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% identical, the identity being identical over the entire length of the nucleic acid of a1) or b1);
d1) at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 with the amino acid sequence encoded by the nucleic acid of a1) or b1) %, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% of the nucleic acid encoding the amino acid sequence, the identity of which is a1) or b1) A nucleic acid that is identical over the entire length of the amino acid sequence encoded by;
e1) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any nucleic acid described in a1) or b1); and
f1) a nucleic acid that encodes the same protein as the nucleic acid of either a1) or b1) but differs from the nucleic acid of a1) or b1) above due to the degeneracy of the genetic code;
A nucleic acid selected from the group consisting of:

The ptsH gene whose activity is reduced in the modified microorganism according to the invention is preferably:
a2) a nucleic acid having the nucleotide sequence of SEQ ID NO: 5;
b2) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6;
c2) a nucleic acid of a2) or b2) and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, A nucleic acid that is at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% identical, the identity being identical over the entire length of the nucleic acid of a2) or b2);
d2) at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 with the amino acid sequence encoded by the nucleic acid of a2) or b2) %, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% identical nucleic acid sequence nucleic acid, the identity of which is a2) or b2) A nucleic acid that is identical over the entire length of the amino acid sequence encoded by;
e2) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any of the nucleic acids described in a2) or b2); and
f2) a nucleic acid that encodes the same protein as the nucleic acid of either a2) or b2), but differs from the nucleic acid of a2) or b2) above due to the degeneracy of the genetic code;
A nucleic acid selected from the group consisting of:

  The term “hybridization” as used herein includes “any process by which a strand of a nucleic acid molecule binds to a complementary strand through base pairing” (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (ie, the strength of pairing between nucleic acid molecules) depends on the degree of complementarity between the nucleic acid molecules, the stringency conditions involved, the Tm of the hybrid formed, and the G in the nucleic acid molecule. : Affected by factors such as C ratio.

  As used herein, the term “Tm” is used to refer to “melting temperature”. The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules is half dissociated into single strands. The equation for calculating the Tm of nucleic acid molecules is well known in the art. As shown in standard references, a simple estimate of the Tm value can be calculated by the formula: Tm = 81.5 + 0.41 (% G + C) when the nucleic acid molecule is in an aqueous solution of 1 M NaCl ( See, for example, Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references include more sophisticated calculations that take structural and sequence features into account for the calculation of Tm. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

  In particular, the term “stringency conditions” refers to a fragment of a complementary nucleic acid molecule (DNA, RNA, ssDNA or ssRNA) or the same 100 or more, 150 or more, 200 or more or 250 or more consecutive nucleotides. Hybridization with 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 ° C., and washing with 2 × SSC, 0.1% SDS at 50 ° C. or 65 ° C., preferably 65 ° C. The condition that hybridizes with a specific nucleic acid molecule (DNA, RNA, ssDNA or ssRNA) under the same conditions as the above. Preferably, the hybridization conditions are: hybridization with 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 ° C., and 1 × SSC at 50 ° C. or 65 ° C., preferably 65 ° C., 0.1 It is equivalent to washing with% SDS, and more preferably, the hybridization conditions are 7% sodium dodecyl sulfate (SDS) at 50 ° C, hybridization with 0.5M NaPO4, 1 mM EDTA, and 50 ° C or 65 ° C. Preferably, it is equivalent to washing with 0.1 × SSC and 0.1% SDS at 65 ° C. Preferably, the complementary nucleotide hybridizes to a fragment or whole of the wcaJ nucleic acid. Alternatively, preferred hybridization conditions are hybridization in 1 × SSC at 65 ° C. or in 1 × SSC and 50% formamide at 42 ° C., followed by washing in 0.3 × SSC at 65 ° C., or 4 at 50 ° C. Includes hybridization in xSSC or 6xSSC and 40% formamide at 40 ° C followed by washing with 2xSSC at 50 ° C. Further preferred hybridization conditions are 0.1% SDS, 0.1 SSD and 65 ° C.

  The ptsA gene and / or ptsH gene or a part of these genes that can be deleted by the “Campbell recombination” or at least one mutation is introduced by the “Campbell recombination” preferably comprises the nucleic acid. .

  The nucleic acids having the nucleotide sequences of SEQ ID NOs: 3 and 5 correspond to the ptsA gene and the ptsH gene of Basfia succinici produce DD1 strain.

According to a further preferred embodiment of the modified microorganism according to the invention, the microorganism is not only due to the reduced activity of the enzyme encoded by the ptsA gene and / or the ptsH gene compared to the wild type,
i) reduced pyruvate formate lyase activity,
ii) reduced lactate dehydrogenase activity,
iii) reduced activity of the enzyme encoded by the wcaJ gene,
iv) reduced activity of the enzyme encoded by the pykA gene,
It is also characterized by at least one of

  Particularly preferred modified microorganisms in this context are the following features or combinations of features: i), ii), iii), iv), i) ii), i) iii), i) iv), ii) iii), ii) iv), iii) iv), i) ii) iii), i) ii) iv), i) iii) iv), ii) iii) iv) and i) ii) iii) iv) There are most preferred here modified microorganisms characterized by features i), ii), iii) and iv).

Modified microorganisms deficient in lactate dehydrogenase and / or deficient in pyruvate formate lyase activity are disclosed in WO-A-2010 / 092155, US2010 / 0159543 and WO-A-2005 / 052135, microorganisms, preferably bacterial cells of the genus Pasteurella These disclosures relating to different approaches for reducing the activity of lactate dehydrogenase and / or pyruvate formate lyase, particularly preferably in the Basphia succinici producers DD1 strain, are incorporated herein by reference. Methods for determining pyruvate formate lyase activity are described, for example, in "Effects of pH and Energy Supply on Activity and Amount of Pyruvate-Formate-Lyase in Streptococcus bovis", Appl. Environ. Microbiol. (2000), Vol. 66, pages 3773-3777, as disclosed in Asanuma N. and Hino T., methods for determining lactate dehydrogenase activity are described, for example, in “Methods of Enzymatic Analysis”, 3 ^rd Edition, Volume III, pages 126-133, In Verlag Chemie, Weinheim, Bergmeyer, HU, Bergmeyer J. and Grassl, M.

  In this context, the reduction of lactate dehydrogenase activity is achieved by inactivation of the ldhA gene (encoding lactate dehydrogenase; LdhA; EC1.1.1.27 or EC1.1.1.28), and the reduction of pyruvate formate lyase is , Activator of pyruvate formate lyase; PflA; encoding EC1.97.1.4) or pflD gene (encoding pyruvate formate lyase; PflD; encoding EC2.3.1.54) Where inactivation of these genes (ie ldhA, pflA and pflD) is preferably by the above “Campbell recombination”, preferably deletion of these genes or parts thereof, Deletion of regulatory elements of at least one gene or at least part thereof, or introduction of at least one mutation into these genes.

  The reduction of the activity of the enzyme encoded by the wcaJ gene is preferably achieved by modification of the wcaJ gene, wherein this genetic modification is preferably performed on the wcaJ gene, such as a deletion of the wcaJ gene or at least a part thereof, a promoter sequence, etc. It is realized by deletion of a regulatory element or at least part thereof or introduction of at least one mutation into the wcaJ gene. In the context of the introduction of at least one mutation into the wcaJ gene, it is particularly preferred that at least one mutation results in the expression of the enzyme encoded by the truncated wcaJ gene. In the truncated enzyme, at least 100 amino acids, preferably at least 125 amino acids, more preferably at least 150 amino acids, and more preferably at least 160 amino acids of the wild type enzyme encoded by the wcaJ gene are deleted from the C-terminus. More preferably. Such a truncated enzyme encoded by the wcaJ gene results in a frameshift mutation, for example by inserting or deleting a nucleotide at the appropriate position of the wcaJ gene, where a stop codon is introduced by this frameshift mutation. Can be obtained. For example, the insertion of a nucleotide between thymine at position 81 encoding lysine and adenine at position 82 results in a frameshift mutation, thereby introducing a stop codon as shown in SEQ ID NO: 13. Such mutations in the wcaJ gene can be introduced, for example, by site-directed or random mutagenesis followed by introduction of the modified gene into the genome of the microorganism by recombination. A mutant of the wcaJ gene can be prepared by mutating SEQ ID NO: 13 of the wcaJ gene sequence by PCR. The “Quickchange Site-directed Mutagenesis Kit (Stratagene)” can be used to perform specific mutagenesis. Random mutagenesis over the entire coding sequence of SEQ ID NO: 13 or only a part thereof can be performed using “Gene Morph II Random Mutagenesis Kit (Stratagene)”.

  Reduction of the activity of the enzyme encoded by the pykA gene is preferably achieved by introduction of at least one mutation into the pykA gene, preferably into the wild-type pykA gene. In this context, the alteration of the nucleic acid sequence of the pykA gene such that at least one mutation differs from the amino acid sequence of the enzyme encoded by the wild type pykA gene by at least one amino acid. It is particularly preferred to provide Mutations to the pykA gene can be introduced, for example, by site-directed or random mutagenesis followed by introduction of the modified gene into the genome of the microorganism by recombination. A mutant of the pykA gene can be prepared by mutating SEQ ID NO: 15 of the gene sequence by PCR. The “Quickchange Site-directed Mutagenesis Kit (Stratagene)” can be used to perform specific mutagenesis. Random mutagenesis over the entire coding sequence of SEQ ID NO: 15 or only a part thereof can be performed using “Gene Morph II Random Mutagenesis Kit (Stratagene)”. The mutation rate is set to the desired amount of mutation through the amount of template DNA used. Multiple mutations are created by targeted combinations of individual mutations or sequential execution of several mutation cycles.

The ldhA gene whose activity can be reduced in the modified microorganism according to the invention is preferably:
α1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 7;
α2) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8;
α3) a nucleic acid of α1) or α2) and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, A nucleic acid that is at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% identical, the identity being identical over the entire length of the nucleic acid α1) or α2);
α4) an amino acid sequence encoded by the nucleic acid of α1) or α2) and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 %, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, and most preferably 100% identical nucleic acids, wherein the nucleic acids have the identity α1) or α2) A nucleic acid that is identical over the entire length of the amino acid sequence encoded by;
α5) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any of the nucleic acids described in α1) or α2); and α6) encoding the same protein as the nucleic acid of either α1) or α2) Is different from the nucleic acid of α1) or α2) above due to the degeneracy of the genetic code;
A nucleic acid selected from the group consisting of:

  The nucleic acid having the nucleotide sequence of SEQ ID NO: 7 corresponds to the ldh gene of Basfiria succinici produce DD1 strain.

The pflA gene whose activity can be reduced in the modified microorganism according to the invention is preferably:
β1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 9;
β2) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 10;
β3) β1) or β2) nucleic acid and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, A nucleic acid that is at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% identical, the identity being the identity over the entire length of the nucleic acid of β1) or β2);
β4) at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 with the amino acid sequence encoded by the nucleic acid of β1) or β2) %, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, and most preferably 100% identical nucleic acids, wherein the nucleic acids have the identity β1) or β2) A nucleic acid that is identical over the entire length of the amino acid sequence encoded by;
β5) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any of the nucleic acids described in β1) or β2); and β6) encoding the same protein as the nucleic acid of either β1) or β2) Is different from the nucleic acid of β1) or β2) above due to the degeneracy of the genetic code;
A nucleic acid selected from the group consisting of:

  The nucleic acid having the nucleotide sequence of SEQ ID NO: 9 corresponds to the pflA gene of Bastia succinici produce DD1 strain.

The pflD gene whose activity can be reduced in the modified microorganism according to the invention is preferably:
γ1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 11;
γ2) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 12;
γ3) γ1) or γ2) with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, A nucleic acid that is at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, most preferably 100% identical, the identity being the identity over the entire length of the nucleic acid of γ1) or γ2);
γ4) an amino acid sequence encoded by the nucleic acid of γ1) or γ2) and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 %, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably nucleic acids encoding amino acid sequences that are 100% identical and having identity γ1) or γ2) A nucleic acid that is identical over the entire length of the amino acid sequence encoded by;
γ5) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any of the nucleic acids described in γ1) or γ2); and γ6) encoding the same protein as the nucleic acid of either γ1) or γ2) Is different from the nucleic acid of γ1) or γ2) due to the degeneracy of the genetic code;
A nucleic acid selected from the group consisting of:

  The nucleic acid having the nucleotide sequence of SEQ ID NO: 11 corresponds to the pflD gene of Basfia succinici produce DD1 strain.

The wcaJ gene whose activity can be reduced in the modified microorganism according to the present invention is preferably:
δ1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 13;
δ2) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 14;
δ3) δ1) or δ2) and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, A nucleic acid that is at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% identical, the identity being the identity over the entire length of the nucleic acid of δ1) or δ2);
δ4) at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 with the amino acid sequence encoded by the nucleic acid of δ1) or δ2) %, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably nucleic acids encoding amino acid sequences that are 100% identical and having identity δ1) or δ2) A nucleic acid that is identical over the entire length of the amino acid sequence encoded by;
δ5) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any of the nucleic acids described in δ1) or δ2); and δ6) encoding the same protein as the nucleic acid of either δ1) or δ2) Is different from the above nucleic acid of δ1) or δ2) due to the degeneracy of the genetic code;
A nucleic acid selected from the group consisting of:

  The nucleic acid having the nucleotide sequence of SEQ ID NO: 13 corresponds to the wcaJ gene of Basfiria succinici produce DD1 strain.

The pykA gene whose activity can be reduced in the modified microorganism according to the invention is preferably:
ε1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 15;
ε2) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 16;
ε3) ε1) or ε2) nucleic acid and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, A nucleic acid that is at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably 100% identical, and whose identity is over the entire length of the nucleic acid of ε1) or ε2);
ε4) at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 with the amino acid sequence encoded by the nucleic acid of ε1) or ε2) %, At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, most preferably nucleic acids encoding amino acid sequences that are 100% identical and having identity ε1) or ε2) A nucleic acid that is identical over the entire length of the amino acid sequence encoded by;
ε5) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any of the nucleic acids described in ε1) or ε2); and ε6) encoding the same protein as the nucleic acid of either ε1) or ε2) Is different from the nucleic acid of ε1) or ε2) above due to the degeneracy of the genetic code;
A nucleic acid selected from the group consisting of:

  The nucleic acid having the nucleotide sequence of SEQ ID NO: 15 corresponds to the pykA gene of the Basfia succinici produce DD1 strain.

In this context, the modified microorganism according to the invention is a genetic modification of A) to E) below:
A) Deletion of the ldhA gene or at least a part thereof, deletion of a regulatory element of the ldhA gene or at least a part thereof, or introduction of at least one mutation into the ldhA gene;
B) a deletion of the pflD gene or at least part thereof, a deletion of a regulatory element of the pflD gene or at least part thereof, or introduction of at least one mutation into the pflD gene; or
deletion of the pflA gene or at least part thereof, deletion of regulatory elements of the pflA gene or at least part thereof, or introduction of at least one mutation into the pflA gene;
C) Deletion of the wcaJ gene or at least part thereof, deletion of a regulatory element of the wcaJ gene or at least part thereof, or introduction of at least one mutation into the wcaJ gene;
D) at least reduce the activity of the enzyme encoded by the pykA gene (least to a reduction), deletion of the pykA gene or at least part thereof, deletion of regulatory elements of the pykA gene or at least part thereof, or pykA gene Introduction of at least one mutation into
E) Deletion of the ptsA gene or at least part thereof, deletion of a regulatory element of the ptsA gene or at least part thereof, or introduction of at least one mutation into the ptsA gene, and / or
a deletion of the ptsH gene or at least part thereof, a deletion of a regulatory element of the ptsH gene or at least part thereof, or introduction of at least one mutation into the ptsH gene;
It is preferable that at least one of these is included.

  Particularly preferred modified microorganisms in this context are the following features or combinations of features: A), B), C), D), E), A) B), A) C), A) D) A) E ), B) C), B) D), B) E), C) D), C) E), D) E), A) B) C), A) B) D), A) B) E), A) C) D), A) C) E), A) D) E), B) C) D), B) C) E), B) D) E) m C) D) E ), A) B) C) D), A) B) C) E), A) B) D) E), A) C) D) E, B) C) D) E) and A) B) Most preferred are modified microorganisms which have C) D) E), characterized here by features A), B), C), D) and E).

According to a first particularly preferred embodiment of the modified microorganism according to the invention, the microorganism is a genetic modification of the following A) to E):
A) Deletion of the ldhA gene or at least part thereof;
B) Deletion of pflA gene or at least part thereof, or deletion of pflD gene or at least part thereof;
C) introduction of at least one mutation into the wcaJ gene that results in the expression of the enzyme encoded by the truncated wcaJ gene;
D) introduction of at least one mutation into the pykA gene that at least reduces the activity of the enzyme encoded by the pykA gene; and
E) Deletion of the ptsA gene or at least part thereof;
including.

According to a second particularly preferred embodiment of the modified microorganism according to the invention, the microorganism is a genetic modification of the following A) to E):
A) Deletion of the ldhA gene or at least part thereof;
B) Deletion of pflA gene or at least part thereof, or deletion of pflD gene or at least part thereof;
C) introduction of at least one mutation into the wcaJ gene that results in the expression of the enzyme encoded by the truncated wcaJ gene;
D) introduction of at least one mutation into the pykA gene that at least reduces the activity of the enzyme encoded by the pykA gene; and
E) Deletion of the ptsH gene or at least part thereof;
including.

Particularly preferred embodiments of modified microorganisms according to the present invention are:
A modified bacterial cell of the Pasteurellaceae family, particularly preferably of the genus Basfia, and even more preferably of Basfia succinici produce.
-Here, preferably by deletion of the ptsA gene, in particular by modification of the ptsA gene which has the nucleic acid sequence of SEQ ID NO: 3 and encodes the enzyme having the amino acid sequence of SEQ ID NO: 4, The activity of the encoded enzyme is reduced;
Here, preferably by modification of the ldhA gene and the pflA gene, in particular by modification of the ldhA gene encoding the LdhA having the nucleic acid sequence of SEQ ID NO: 7 and having the amino acid sequence of SEQ ID NO: 8, and The nucleic acid described in SEQ ID NO: 9, having the nucleic acid sequence described in SEQ ID NO: 9, and by modifying the pflA gene encoding PflA having the amino acid sequence described in SEQ ID NO: 10, or by modifying the ldhA gene and the pflD gene PflD having the sequence and having the nucleic acid sequence set forth in SEQ ID NO: 12 by modification of the ldhA gene encoding LdhA having the amino acid sequence set forth in SEQ ID NO: 8 and the amino acid sequence set forth in SEQ ID NO: 12 Modification of the encoding pflD gene reduces the activity of lactate dehydrogenase and pyruvate formate lyase;
-Where the wcaJ gene or at least a portion thereof is deleted, or at least one mutation has the wcaJ gene, in particular the nucleic acid sequence set forth in SEQ ID NO: 13, and the amino acid sequence set forth in SEQ ID NO: 14 Wherein the at least one mutation is preferably at least 100 amino acids, preferably at least 125 amino acids, more preferably at least 150 amino acids of the wild-type enzyme encoded by the wcaJ gene, and More preferably, at least 160 amino acids result in expression of the enzyme deleted from the C-terminus;
And wherein at least one mutation has been introduced into the pykA gene, in particular the pykA gene having the nucleic acid sequence set forth in SEQ ID NO: 15 and encoding the protein having the amino acid sequence set forth in SEQ ID NO: 16, preferably Wherein at least one mutation results in a substitution of at least one amino acid of the enzyme encoded by the pykA gene, most preferably the mutation is a substitution of glycine by cysteine at position 167 or position 417 in the enzyme encoded by the pykA gene Cysteine substitution at position 171 or alanine glycine at position 171 or glycine substitution at position 167 and cysteine substitution at position 417 and tyrosine substitution at position 417, or glycine substitution at position 167 and alanine at position 171 Replacement with glycine Or substitution of a glycine with alanine in the substituted and 171 of by tyrosine cysteine at 417 position, or position 167 substitution of a glycine with cysteines in, resulting in at least a substitution of glycine alanine in the substituted and 171 of by tyrosine cysteine in 417 of.
A modified bacterial cell of the Pasteurellaceae family, particularly preferably of the genus Basfia, and even more preferably of Basfia succinici produce.
-Here, preferably by deletion of the ptsH gene, in particular by modification of the ptsH gene having the nucleic acid sequence of SEQ ID NO: 5 and encoding the enzyme having the amino acid sequence of SEQ ID NO: 6, The activity of the encoded enzyme is reduced;
Here, preferably by modification of the ldhA gene and the pflA gene, in particular by modification of the ldhA gene encoding the LdhA having the nucleic acid sequence of SEQ ID NO: 7 and having the amino acid sequence of SEQ ID NO: 8, and The nucleic acid described in SEQ ID NO: 9, having the nucleic acid sequence described in SEQ ID NO: 9, and by modifying the pflA gene encoding PflA having the amino acid sequence described in SEQ ID NO: 10, or by modifying the ldhA gene and the pflD gene PflD having the sequence and having the nucleic acid sequence set forth in SEQ ID NO: 12 by modification of the ldhA gene encoding LdhA having the amino acid sequence set forth in SEQ ID NO: 8 and the amino acid sequence set forth in SEQ ID NO: 12 Modification of the encoding pflD gene reduces the activity of lactate dehydrogenase and pyruvate formate lyase;
-Here, the wcaJ gene, or at least a part thereof is deleted, or at least one mutation has the wcaJ gene, particularly the nucleic acid sequence set forth in SEQ ID NO: 13, and the amino acid sequence set forth in SEQ ID NO: 14. Wherein at least one mutation is preferably at least 100 amino acids, preferably at least 125 amino acids, more preferably at least 150 amino acids of the wild-type enzyme encoded by the wcaJ gene, And more preferably at least 160 amino acids result in expression of the enzyme deleted from the C-terminus;
And wherein at least one mutation has been introduced into the pykA gene, in particular the pykA gene having the nucleic acid sequence set forth in SEQ ID NO: 15 and encoding the protein having the amino acid sequence set forth in SEQ ID NO: 16, preferably Wherein at least one mutation results in a substitution of at least one amino acid of the enzyme encoded by the pykA gene, most preferably the mutation is a substitution of glycine by cysteine at position 167 or position 417 in the enzyme encoded by the pykA gene Cysteine substitution at position 171 or alanine glycine at position 171 or glycine substitution at position 167 and cysteine substitution at position 417 and tyrosine substitution at position 417, or glycine substitution at position 167 and alanine at position 171 Replacement with glycine Or substitution of a glycine with alanine in the substituted and 171 of by tyrosine cysteine at 417 position, or position 167 substitution of a glycine with cysteines in, resulting in at least a substitution of glycine alanine in the substituted and 171 of by tyrosine cysteine in 417 of.

The contributions to solving the first mentioned issue are further:
I) culturing a modified microorganism according to the invention in a culture medium comprising at least one assimilable carbon source that allows the modified microorganism to produce organic compounds, thereby obtaining a fermentation broth comprising the organic compounds;
II) Provided by a method for producing an organic compound comprising the step of recovering the organic compound from the fermentation broth obtained in process step I).

  In process step I), the modified microorganism according to the invention is cultivated in a culture medium comprising at least one assimilable carbon source that enables the modified microorganism to produce organic compounds, and thereby a fermentation broth comprising organic compounds. Is obtained. Preferred organic compounds that can be produced by the process according to the invention are carboxylic acids such as formic acid, lactic acid, propionic acid, 2-hydroxypropionic acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, acrylic acid, pyruvic acid or their carboxylic acids. Acid salts, dicarboxylic acids such as malonic acid, succinic acid, malic acid, tartaric acid, glutaric acid, itaconic acid, adipic acid or salts thereof, tricarboxylic acids such as citric acid or salts thereof, alcohols such as methanol or ethanol, amino acids For example, L-asparagine, L-aspartic acid, L-arginine, L-isoleucine, L-glycine, L-glutamine, L-glutamic acid, L-cysteine, L-serine, L-tyrosine, L-tryptophan, L-threonine , L-valine, L-histidine, L-proline, L-methionine, L-lysine, L-leucine Including the.

According to a preferred embodiment of the process according to the invention, the organic compound is succinic acid. The term “succinic acid” as used in the context of the present invention is to be understood in the broadest sense and its salts (ie succinates), eg alkali metal salts such as Na ⁺ and K ⁺ salts of succinic acid or Mg ^{2 Also included} are alkaline earth metal salts such as ⁺ and Ca ²⁺ salts or ammonium salts or anhydrides.

  The modified microorganism according to the invention is preferably incubated in the culture medium at a temperature in the range of about 10-60 ° C or 20-50 ° C or 30-45 ° C and at a pH of 5.0-9.0 or 5.5-8.0 or 6.0-7.0. .

  Preferably, the organic compound, especially succinic acid, is produced under anaerobic conditions. Anaerobic conditions can be achieved by deaerating the components of the reaction medium by conventional techniques, for example by introducing carbon dioxide or nitrogen or mixtures thereof and optionally hydrogen, for example at a flow rate of 0.1-1 or 0.2-0.5 vvm. It can be established by maintaining conditions. Aerobic conditions can be established by conventional techniques, for example by introducing air or oxygen, for example at a flow rate of 0.1-1 or 0.2-0.5 vvm. Where appropriate, a slight excess pressure of 0.1 to 1.5 bar may be applied in the process.

  The assimilable carbon source is preferably sucrose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, D-fructose, D-glucose, D-xylose, L-arabinose, Selected from the group consisting of D-galactose, D-mannose, glycerol and mixtures thereof, or a composition comprising at least one of the aforementioned compounds, or selected from degradation products of starch, cellulose, hemicellulose and / or lignocellulose Is done. A preferred assimilable carbon source is sucrose. Further preferred mixtures are mixtures of sucrose and at least one further assimilable carbon source, such as sucrose and maltose, sucrose and D-fructose, sucrose and D-glucose, sucrose and D-xylose, sucrose and It is a mixture of L-arabinose, sucrose and D-galactose, sucrose and D-mannose.

  According to a preferred embodiment of the process according to the invention, based on the total weight of the assimilable carbon source excluding carbon dioxide, at least 50% by weight of the assimilable carbon source, preferably at least 75% by weight, more preferably at least 90% by weight, even more preferably at least 95% by weight, and most preferably at least 99% by weight is sucrose.

The initial concentration of the assimilable carbon source, preferably the initial concentration of sucrose, is preferably adjusted to a value in the range of 5-100 g / l, preferably 5-75 g / l and more preferably 5-50 g / l, The range can be maintained during the culture. The pH of the reaction medium is adjusted to a suitable base such as gaseous ammonia, NH ₄ HCO ₃ , (NH ₄ ) ₂ CO ₃ , NaOH, Na ₂ CO ₃ , NaHCO ₃ , KOH, K ₂ CO ₃ , KHCO ₃ , Mg (OH ) ₂ , MgCO ₃ , Mg (HCO ₃ ) ₂ , Ca (OH) ₂ , CaCO ₃ , Ca (HCO ₃ ) ₂ , CaO, CH ₆ N ₂ O ₂ , C ₂ H ₇ N and / or mixtures thereof It can be controlled by addition. These alkali neutralizers are particularly needed when the organic compound formed in the course of the fermentation process is a carboxylic acid or dicarboxylic acid. When the organic compound is succinic acid, Mg (OH) ₂ is a particularly preferred base.

  The fermentation step I) according to the invention can be carried out, for example, in a stirred fermenter, bubble column and loop reactor. A comprehensive overview of possible process types and geometric designs including agitation types can be found in Chmiel, Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Volume 1. In the process according to the invention, typical variants available are known or explained to those skilled in the art, e.g. the following variants of Chmiel, Hammes and Bailey: Biochemical Engineering, e.g. with or without recycling of biomass, Batch, fed-batch, repetitive fed batch or other continuous culture. Depending on the production strain, injection of air, oxygen, carbon dioxide, hydrogen, nitrogen or a suitable gas mixture can be performed to achieve good yield (YP / S).

In process step I), particularly preferred conditions for producing organic acids, in particular succinic acid, are:
Assimitable carbon source: Sucrose temperature: 30-45 ° C
pH: 5.5-7.0
Supply gas: CO ₂
It is.

  In process step I), an assimilable carbon source, preferably sucrose, is converted to an organic compound, preferably succinic acid, with a carbon yield YP / S of at least 0.5 g / g and up to about 1.18 g / g; 6g / g, at least 0.7g / g, at least 0.75g / g, at least 0.8g / g, at least 0.85g / g, at least 0.9g / g, at least 0.95g / g, at least 1.0g / g, at least 1.05g More preferably, it is converted with a carbon yield YP / S of at least 1.1 g / g (organic compound / carbon, preferably succinic acid / carbon).

In process step I), an assimilable carbon source, preferably sucrose, is converted to an organic compound, preferably succinic acid, at least 0.6 gg DCW ^-1 h ^-1 organic compound, preferably succinic acid, or at least 0.65 gg DCW ^-1 h ^-1, at least 0.7gg DCW ^-1 h ^-1, at least 0.75gg DCW ^-1 h ^-1, or at least 0.77 gg DCW ^-1 h ^-1 organic compounds, preferably organic compounds specific productivity yield of succinic acid, More preferably, it is more preferably converted to succinic acid.

  In process step I), the assimilable carbon source, preferably sucrose, is at least 2.2 g / (L × h) or at least 2.5 g / (L × h), at least 2.75 g / of organic compounds, preferably succinic acid. (L × h), at least 3 g / (L × h), at least 3.25 g / (L × h), at least 3.5 g / (L × h), at least 3.7 g / (L × h), at least 4.0 g / ( L × h), at least 4.5 g / (L × h) or at least 5.0 g / (L × h) organic compound, preferably succinic acid in space-time yield, converted to organic compound, preferably succinic acid. Is more preferable. According to another preferred embodiment of the process according to the invention, in process step I) the modified microorganism is at least 20 g / L, more preferably at least 25 g / l and even more preferably at least 30 g / l assimilable carbon. The source, preferably sucrose, is converted to at least 20 g / l, more preferably at least 25 g / l and even more preferably at least 30 g / l of organic compound, preferably succinic acid.

The different yield parameters described herein (“carbon yield” or “YP / S”; “specific productivity yield”; or “space-time yield (STY)”) are well known in the art. For example as described by Song and Lee, 2006. “Carbon yield” and “YP / S” (respectively expressed as mass of organic compound produced / mass of assimilable carbon source consumed) are used synonymously herein. Specific productivity yield describes the amount of product such as L fermentation broth per g dry biomass and succinic acid produced per h. Dry cell weight, described as “DCW”, describes the amount of biologically active microorganism in the biochemical reaction. This value is expressed as g product per gDCW per h (ie ggDCW ⁻¹ h ⁻¹ ). Space time yield (STY) is defined as the ratio of the total amount of organic compounds formed in the fermentation process to the volume of the culture over the entire time of culture. The space-time yield is also known as “volumetric productivity”.

  In process step II), the organic compound is recovered from the fermentation broth obtained in process step I).

  Usually, the recovery process involves separating the recombinant microorganisms from the fermentation broth as so-called “biomass”. Processes for removing biomass are known to those skilled in the art and include filtration, sedimentation, flotation or combinations thereof. As a result, biomass can be removed using, for example, a centrifuge, separator, decanter, filter, or in a flotation device. For maximum recovery of valuable products, it is often desirable to wash the biomass, for example in the form of diafiltration. The choice of method also depends on the biomass content and nature of the fermentation broth and the interaction of the biomass with organic compounds (ie valuable products). In one embodiment, the fermentation broth can be sterilized or pasteurized. In a further embodiment, the fermentation broth is concentrated. Depending on the requirements, this concentration can be carried out batchwise or continuously. The pressure and temperature range should be selected so that, first, the use of minimal equipment and energy is required so that no product damage occurs. A skilled selection of pressure and temperature levels, especially for multi-stage evaporation, allows energy conservation.

  The recovery process may further comprise an additional purification step in which the organic compound, preferably succinic acid, is further purified. However, when the organic compound is converted to a secondary organic product by a chemical reaction as described below, further purification of the organic compound is not necessarily required depending on the type of reaction and the reaction conditions. For the purification of the organic compounds obtained in process step II), preferably for the purification of succinic acid, methods known to the person skilled in the art, for example crystallization, filtration, electrodialysis and chromatography can be used. When the organic compound is succinic acid, for example, succinic acid is precipitated by using succinic acid as a calcium succinate product by using hydroxylation, oxidation, calcium carbonate or bicarbonate for neutralization. Can be isolated by filtration. Succinic acid is recovered from the precipitated calcium succinate product by acidification with sulfuric acid followed by filtration to remove precipitated calcium sulfate (gypsum). The resulting solution can be further purified by ion exchange chromatography to remove unwanted residual ions. Alternatively, if magnesium hydroxide, magnesium carbonate, or a mixture thereof is used to neutralize the fermentation broth, the fermentation broth obtained in process step I) will convert the magnesium succinate contained in the medium to the acid form (ie This acid form can then be crystallized by cooling the acidified medium. Examples of further suitable purification processes are EP-A-1005562, WO-A-2008 / 010373, WO-A-2011 / 082378, WO-A-2011 / 043443, WO-A-2005 / 030973, WO-A -2011/123268 and WO-A-2011 / 064151 and EP-A-2360137.

According to a preferred embodiment of the process according to the invention, the process further comprises process steps:
III) Conversion of the organic compound contained in the fermentation broth obtained in process step I) to a secondary organic product different from the organic compound by at least one chemical reaction, or recovered organic obtained in process step II) Includes conversion of compounds.

  When the organic compound is succinic acid, preferred secondary organic products consist of succinic acid ester and its polymer, tetrahydrofuran (THF), 1,4-butanediol (BDO), γ-butyrolactone (GBL) and pyrrolidone. Selected from the group.

According to a preferred embodiment for the production of THF, BDO and / or GBL, the process is:
b1) Direct catalytic hydrogenation of the succinic acid obtained in process step I) or II) to THF and / or BDO and / or GBL, or
b2) Chemical esterification of the succinic acid and / or succinate obtained in process step I) or II) to the corresponding di-lower alkyl ester, followed by THF and / or BDO and / or GBL of said ester Catalytic hydrogenation,
One of these.

According to a preferred embodiment for the production of pyrrolidone, the process is:
b) Chemical conversion of the ammonium succinate salt obtained in process step I) or II) to pyrrolidone in a manner known per se.

  For details of the preparation of these compounds, see US-A-2010 / 0159543 and WO-A-2010 / 092155.

The contribution to solving the initially described problem is further provided by the use of the modified microorganism according to the invention for the fermentative production of organic compounds. Preferred organic compounds are those already described for the process according to the invention, succinic acid being the most preferred organic compound. Furthermore, preferred conditions for the fermentative production of organic compounds, preferably succinic acid, are those already described for process step I) of the process according to the invention. A preferred assimilable carbon source used for fermentative production of organic compounds, particularly for succinic acid, is sucrose.
Various embodiments of the invention are described below.
1. Compared to the wild type,
-Reduced activity of the enzyme encoded by the ptsA gene,
Reduced activity of the enzyme encoded by the ptsH gene, or
-Reduced activity of the enzyme encoded by the ptsA gene and reduced activity of the enzyme encoded by the ptsH gene
The modified microorganism according to claim 1, wherein the wild type from which the modified microorganism is derived belongs to the family Pasteurellaceae.
2. 2. The modified microorganism according to 1 above, wherein the wild type from which the modified microorganism is derived belongs to the genus Basfia.
3. The ptsA gene is:
a1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 3;
b1) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 4;
c1) a nucleic acid that is at least 70% identical to the nucleic acid of a1) or b1), wherein the identity is identical over the entire length of the nucleic acid of a1) or b1);
d1) a nucleic acid that encodes an amino acid sequence that is at least 70% identical to the amino acid sequence encoded by the nucleic acid of a1) or b1), wherein the identity is the full length of the amino acid sequence encoded by the nucleic acid of a1) or b1) Nucleic acids that are over the same identity;
e1) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any nucleic acid described in a1) or b1); and
f1) a nucleic acid that encodes the same protein as the nucleic acid of either a1) or b1) but differs from the nucleic acid of a1) or b1) above due to the degeneracy of the genetic code;
3. The modified microorganism according to 1 or 2 above, which comprises a nucleic acid selected from the group consisting of:
4). The ptsH gene is:
a2) a nucleic acid having the nucleotide sequence of SEQ ID NO: 5;
b2) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6;
c2) a nucleic acid that is at least 70% identical to the nucleic acid of a2) or b2) and whose identity spans the entire length of the nucleic acid of a2) or b2);
d2) a nucleic acid that encodes an amino acid sequence that is at least 70% identical to the amino acid sequence encoded by the nucleic acid of a2) or b2), the identity of which is the full length of the amino acid sequence encoded by the nucleic acid of a2) or b2) Nucleic acids that are over the same identity;
e2) a nucleic acid capable of hybridizing under stringent conditions to the complementary sequence of any of the nucleic acids described in a2) or b2); and
f2) a nucleic acid that encodes the same protein as the nucleic acid of either a2) or b2), but differs from the nucleic acid of a2) or b2) above due to the degeneracy of the genetic code;
3. The modified microorganism according to 1 or 2 above, which comprises a nucleic acid selected from the group consisting of:
5. Compared to the wild type, the microorganism has the following characteristics:
i) reduced pyruvate formate lyase activity,
ii) reduced lactate dehydrogenase activity,
iii) reduced activity of the enzyme encoded by the wcaJ gene,
iv) reduced activity of the enzyme encoded by the pykA gene,
The modified microorganism according to any one of 1 to 4, further comprising at least one of the following.
6). The microorganism is genetically modified in the following A) to E):
A) Deletion of the ldhA gene or at least a part thereof, deletion of a regulatory element of the ldhA gene or at least a part thereof, or introduction of at least one mutation into the ldhA gene;
B) a deletion of the pflD gene or at least part thereof, a deletion of a regulatory element of the pflD gene or at least part thereof, or introduction of at least one mutation into the pflD gene; or
deletion of the pflA gene or at least part thereof, deletion of regulatory elements of the pflA gene or at least part thereof, or introduction of at least one mutation into the pflA gene;
C) Deletion of the wcaJ gene or at least part thereof, deletion of a regulatory element of the wcaJ gene or at least part thereof, or introduction of at least one mutation into the wcaJ gene;
D) at least one deletion of the pykA gene or at least a part thereof, a deletion of a regulatory element of the pykA gene or at least a part thereof, or at least one mutation to the pykA gene, which at least reduces the activity of the enzyme encoded by the pykA gene Introduction of;
E) Deletion of the ptsA gene or at least part thereof, deletion of a regulatory element of the ptsA gene or at least part thereof, or introduction of at least one mutation into the ptsA gene, and / or
a deletion of the ptsH gene or at least part thereof, a deletion of a regulatory element of the ptsH gene or at least part thereof, or introduction of at least one mutation into the ptsH gene;
6. The modified microorganism according to 5 above, comprising at least one of
7). The microorganism is:
A) Deletion of the ldhA gene or at least part thereof;
B) Deletion of pflA gene or at least part thereof, or deletion of pflD gene or at least part thereof;
C) introduction of at least one mutation into the wcaJ gene that results in the expression of the enzyme encoded by the truncated wcaJ gene;
D) introduction of at least one mutation into the pykA gene; and
E) Deletion of the ptsA gene or at least part thereof;
7. The modified microorganism according to 6 above, comprising
8). The microorganism is:
A) Deletion of the ldhA gene or at least part thereof;
B) Deletion of pflA gene or at least part thereof, or deletion of pflD gene or at least part thereof;
C) introduction of at least one mutation into the wcaJ gene that results in the expression of the enzyme encoded by the truncated wcaJ gene;
D) introduction of at least one mutation into the pykA gene; and
E) Deletion of the ptsH gene or at least part thereof;
7. The modified microorganism according to 6 above, comprising
9. I) culturing the modified microorganism according to any one of 1 to 8 above in a culture medium comprising at least one assimilable carbon source that enables the modified microorganism to produce an organic compound, whereby the organic compound is Obtaining a fermentation broth comprising;
II) Step of recovering organic compounds from the fermentation broth obtained in process step I)
A method for producing an organic compound, comprising:
10. 10. The method according to 9 above, wherein the organic compound is succinic acid.
11. 11. The method according to 9 or 10 above, wherein at least 50% by weight of the assimilable carbon source is sucrose, based on the total weight of the assimilable carbon source excluding carbon dioxide.
12 The method comprises process steps:
III) Conversion of organic compounds contained in the fermentation broth obtained in process step I) to secondary organic products different from said organic compounds by at least one chemical reaction, or recovered organic obtained in process step II) Compound conversion
The method according to any one of 9 to 11, further comprising:
13. The organic compound is succinic acid and the secondary organic product is selected from the group consisting of succinic acid esters or polymers thereof, tetrahydrofuran (THF), 1,4-butanediol (BDO), γ-butyrolactone (GBL) and pyrrolidone 13. The method according to 12 above.
14 Use of the modified microorganism according to any one of the above 1 to 8 for fermentative production of an organic compound.
15. 15. Use according to 14 above, wherein the organic compound is succinic acid and sucrose is used as an assimilable carbon source.

  The invention is explained in more detail below using figures and non-limiting examples.

Example 1: General method of transformation of Basphia succinici produce
Bastia succinici produce DD1 (wild type) was transformed into DNA by electroporation using the following protocol:
To prepare the preculture, DD1 was inoculated from frozen stock into 40 ml BHI (Brain Heart Infusion, Becton, Dickinson and Company) in a 100 ml shake flask. Incubation was performed overnight at 37 ° C; 200 rpm. To prepare the main culture, 100 ml of BHI was placed in a 250 ml shake flask and inoculated with the preculture at a final OD (600 nm) of 0.2. Incubation was performed at 37 ° C. and 200 rpm. Cells were harvested at an OD of approximately 0.5, 0.6, and 0.7 and the pellet was washed once with 10% cold glycerol at 4 ° C. and resuspended in 2 ml 10% glycerol (4 ° C.).

  100 μl of competent cells were mixed with 2-8 μg of plasmid DNA and kept in an electroporation cuvette with a width of 0.2 cm for 2 minutes on ice. Electroporation is performed under the following conditions: 400Ω; 25 μF; 2.5 kV (Gene Pulser, Bio-Rad). 1 ml of chilled BHJ was added immediately after electroporation and incubation was carried out at 37 ° C. for about 2 h.

  Cells were seeded in BHI containing 5 mg / L chloramphenicol and incubated at 37 ° C. for 2-5 days until transformant colonies were visualized. Clones were isolated and re-streaked to BHI containing 5 mg / L chloramphenicol until clone purity was obtained.

Example 2:
a) Preparation of deletion construct The deletion plasmid was constructed based on the pSacB vector (SEQ ID NO: 17). FIG. 1 shows a schematic map of the pSacB plasmid. The 5 ′ and 3 ′ flanking regions (approximately 1500 bp each) of the chromosomal fragment to be deleted are amplified by PCR from the chromosomal DNA of Basfia succinici produce and introduced into the vector using standard methods. did. Usually, at least 80% of the ORF was targeted for deletion. Such methods include a deletion plasmid for lactate dehydrogenase ldhA, pSacB_delta_ldhA (SEQ ID NO: 18), a deletion plasmid for pyruvate formate lyase activating enzyme pflA, pSacB_delta_pflA (SEQ ID NO: 19), ptsA gene, psacB_delta_ptsA (SEQ ID NO: 22), and the ptsH gene, psacB_delta_ptsH (SEQ ID NO: 23). 2, 3, 6 and 7 show schematic maps of the pSacB_delta_ldhA, pSacB_delta_pflA, psacB_delta_ptsA and psacB_delta_ptsH plasmids, respectively.

  In the plasmid sequence of pSacB (SEQ ID NO: 17), the sacB gene is contained at bases 2380-3801. The sacB promoter is contained at bases 3802-4264. The chloramphenicol gene is contained at bases 526-984. The origin of replication of E. coli (ori EC) is contained in bases 1477-2337 (see FIG. 1).

  In the plasmid sequence of pSacB_delta_ldhA (SEQ ID NO: 18), the 5 'flanking region of the ldhA gene that is homologous to the genome of Basfia succinici produce is contained at bases 1519-2850, whereas Basfia succinici produce The 3 ′ flanking region of the ldhA gene that is homologous to the genome is contained at bases 62-1518. The sacB gene is contained in bases 5169-6590. The sacB promoter is contained at bases 6651 to 7053. The chloramphenicol gene is contained at bases 3315-3773. The origin of replication of E. coli (ori EC) is contained in bases 4266-5126 (see FIG. 2).

  In the plasmid sequence of pSacB_delta_pflA (SEQ ID NO: 19), the 5 'flanking region of the pflA gene that is homologous to the genome of Basfia succinici produce is contained at bases 1506 to 3005, whereas Basfia succinici produce The 3 ′ flanking region of the pflA gene that is homologous to the genome is contained in bases 6 to 1505. The sacB gene is contained at bases 5278-6699. The sacB promoter is contained at bases 6700-7162. The chloramphenicol gene is contained at bases 3424-3882. The origin of replication of E. coli (ori EC) is contained in bases 4375-5235 (see FIG. 3).

  In the plasmid sequence of pSacB_delta_ptsA (SEQ ID NO: 22), the 5 ′ flanking region of the ptsA gene that is homologous to the genome of Basfia succinici produce is contained at bases 1506 to 3005, whereas the Basfia succinici producers The 3 ′ flanking region of the ptsA gene that is homologous to the genome is contained in bases 6 to 1505. The sacB gene is contained at bases 5278-6699. The sacB promoter is contained at bases 6700-7162. The chloramphenicol gene is contained at bases 3424-3882. The origin of replication of E. coli (ori EC) is contained in bases 4375-5235 (see FIG. 6).

In the plasmid sequence of pSacB_delta_ptsH (SEQ ID NO: 23), the 5 ′ flanking region of the ptsH gene that is homologous to the genome of Basfia succinici produce is contained at bases 1541-3055, whereas the Basfia succinici producers The 3 ′ flanking region of the ptsH gene that is homologous to the genome is contained in bases 6 to 1540. The sacB gene is contained in bases 5328 to 6749. The sacB promoter is contained at bases 6750-7122. The chloramphenicol gene is contained at bases 3474-3932. The origin of replication of E. coli (ori EC) is contained in bases 4425-5285 (see FIG. 7).
b) Preparation of constructs used for introduction of point mutations into the pykA gene and wcaJ gene
In the plasmid sequence of pSacB_pykA1 (SEQ ID NO: 20), a part of the pykA gene that is homologous to the genome of Basfia succinici produce is contained in bases 6 to 1185. The sacB gene is contained in bases 3458-4879. The sacB promoter is contained at bases 4880-5342. The chloramphenicol gene is contained at bases 1604-2062. The origin of replication of E. coli (ori EC) is contained at bases 2555-3415 (see FIG. 4). Plasmid pSacB_pykA1 introduces a G to T mutation in the pykA gene that ultimately results in the replacement of G (glycine) at position 167 in the PykA protein (SEQ ID NO: 16) with C (cysteine).

In the plasmid sequence of pSacB_wcaJ ^* (SEQ ID NO: 21), the 5 'flanking region of the wcaJ gene that is homologous to the genome of Basfia succinici produce is contained at bases 1838-3379, while Basfia succinici produce The 3 'flanking region of the wcaJ gene that is homologous to the genome of is contained in bases 6-1236. The sacB gene is contained in bases 5562-7073. The sacB promoter is contained at bases 7074-7536. The chloramphenicol gene is contained in bases 3798-4256. The origin of replication of E. coli (ori EC) is contained in bases 4749-5609. The wcaJ gene is contained at bases 1237-1837 with an insertion of a lysine-encoding nucleotide between thymine at position 81 and adenine at position 82 (corresponding to position 1756 of pSacB_wcaJ ^* , see FIG. 5). This insertion results in a frameshift mutation, and a stop codon is introduced that results in the expression of the enzyme truncated by this frameshift mutation.

Example 3: Production of improved succinate production strain
a) Basfia succinici produce DD1 was transformed with pSacB_delta_ldhA as described above and “Campbell in” to obtain a “Campbell in” strain. Transformation and integration of the Basfia succinici produce into the genome was confirmed by the band obtained by PCR of the plasmid integration event into the Basfia succinici produce genome.

  Thereafter, the “Campbell in” strain was “Campbell out” using an agar plate containing sucrose as a counter selection medium to select for (functional) deletion of the sacB gene. Therefore, the “Campbell in” strain was incubated overnight at 37 ° C. and 220 rpm in 25-35 ml of non-selective medium (BHI without antibiotics). The overnight culture was then streaked onto a BHI plate containing freshly prepared sucrose (10%, no antibiotics) and incubated overnight at 37 ° C. (“first sucrose transfer”). Single colonies obtained from the first transfer were streaked again on freshly prepared (10%) BHI plates containing sucrose and incubated overnight at 37 ° C. (“second sucrose transfer”). This procedure was repeated until a minimum of 5 transitions in sucrose was completed ("third, fourth, fifth sucrose transition"). The term “first to fifth sucrose transition” refers to sucrose and sucrose after chromosomal integration of a vector containing the sacB-levan-sucrase gene to select strains having deletions of the sacB gene and surrounding plasmid sequences. Refers to the transfer of strains to agar plates containing growth media. Single colonies from the fifth transfer plate were inoculated into 25-35 ml non-selective medium (BHI without antibiotics) and cultured overnight at 37 ° C., 220 rpm. Overnight cultures were serially diluted and seeded on BHI plates to obtain isolated single colonies.

  A “Campbell out” strain containing a mutation / deletion of the ldhA gene was confirmed by chloramphenicol sensitivity. Mutation / deletion mutants in these strains were identified and confirmed by PCR analysis. As a result, an ldhA deletion mutant Basfia succinici produce DD1ΔldhA was obtained.

  b) Basfia succinici producers ΔldhA was transformed with the above pSacB_delta_pflA and “Campbell in” to obtain a “Campbell in” strain. Transformation and integration was confirmed by PCR. Thereafter, the “Campbell in” strain was “Campbell out” as described above. Deletion mutants in these strains were identified and confirmed by PCR analysis. As a result, the ldhA pflA double deletion mutant Basfia succinici produce DD1ΔldhAΔpflA was obtained.

  c) Basfia succinici producers ΔldhAΔpflA was transformed with the above pSacB_pykA1 and “Campbell in” to obtain a “Campbell in” strain. Thereafter, the “Campbell in” strain was “Campbell out” as described above. Deletion mutants in these strains were identified and confirmed by PCR analysis. This resulted in mutant Basfia succinici produce DD1ΔldhAΔpflA pykA1 expressing pyruvate kinase in which ldhA and pflA were deleted and glycine at amino acid position 167 was replaced by cysteine.

d) Basfia succinici produce ∆ldhA∆pflA pykA1 was transformed with the above pSacB_wcaJ ^* and "Campbell in" to obtain a "Campbell in" strain. Thereafter, the “Campbell in” strain was “Campbell out” as described above. Deletion mutants in these strains were identified and confirmed by PCR analysis. This results in a mutant Basfia succinici product that expresses pyruvate kinase in which ldhA and pflA are deleted, glycine at amino acid position 167 is replaced by cysteine, and expresses the enzyme encoded by the truncated wcaJ gene. Sense DD1ΔldhAΔpflA pykA1 wcaJ ^* was obtained.

e) Basfia succinici producers ΔldhAΔpflA pykA1 wcaJ ^* was transformed with the above pSacB_ptsA and “Campbell in” was obtained to obtain a “Campbell in” strain. Transformation and integration was confirmed by PCR. Thereafter, the “Campbell in” strain was “Campbell out” as described above. Deletion mutants in these strains were identified and confirmed by PCR analysis. As a result, the mutant Basfia succinici productions DD1ΔldhAΔpflA pykA1 wcaJ ^* ΔptsA was obtained.

f) Basfia succinici producers ΔldhAΔpflA pykA1 wcaJ ^* was transformed with the above pSacB_hpR and “Campbell in” to obtain a “Campbell in” strain. Transformation and integration was confirmed by PCR. Thereafter, the “Campbell in” strain was “Campbell out” as described above. Deletion mutants in these strains were identified and confirmed by PCR analysis. As a result, the mutant Basfia succinici productions DD1ΔldhAΔpflApykA1 wcaJ ^* ΔptsH was obtained.

Example 4: Culture of various DD1 strains in glucose and sucrose Productivity was analyzed using the following media and incubation conditions.

1. Medium Preparation The composition and preparation of the culture medium CGM are as described in Table 2 below.
The composition and preparation of the culture medium LSM_3 is as described in Tables 3, 4, and 5 below.

2. Cultivation and analysis For pre-culture growth, 50 ml of the listed in Table 2 was used with freshly grown bacteria on BHI agar plates (incubated overnight at 37 ° C under anaerobic conditions) in a CO ₂ atmosphere. A 100 ml serum bottle containing CGM liquid medium and having an airtight butyl rubber stopper was inoculated at OD600 = 0.75. The bottles were incubated at 37 ° C. and 170 rpm (shaking diameter: 2.5 cm). For main culture growth, use 2.5 ml bacterial culture in CGM medium (after 10 h incubation), and contain 50 mL of LSM_3 liquid medium listed in Table 5 under CO ₂ atmosphere, and airtight butyl rubber stopper 100 ml serum bottles with Succinic acid production was quantified via HPLC (HPLC methods are described in Tables 7 and 8). Cell proliferation was measured by measuring absorbance at 600 nm (OD600) using a spectrophotometer (Ultrospec 3000, Amersham Biosciences, Uppsala Sweden).

3. Results Table 6 shows the results of various DD1 strain culture experiments.

Array

_{Accession Number:} DSM 18541

Claims (10)

A modified microorganism of the Pasteurellaceae family having reduced activity of the enzyme encoded by the ptsA gene compared to the wild type, wherein the reduction of the activity of the enzyme encoded by the ptsA gene is achieved by modification of the ptsA gene, The gene is:
a1) a nucleic acid having the nucleotide sequence of SEQ ID NO: 3;
b1) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 4;
c1) a nucleic acid that is at least 90 % identical to the nucleic acid of a1) or b1), wherein the identity is identical over the entire length of the nucleic acid of a1) or b1);
d1) a nucleic acid encoding an amino acid sequence that is at least 90 % identical to the amino acid sequence encoded by the nucleic acid of a1) or b1), wherein the identity is the full length of the amino acid sequence encoded by the nucleic acid of a1) or b1) Nucleic acids that are over the identity; and
e1) a nucleic acid that encodes the same protein as the nucleic acid of either a1) or b1), but differs from the nucleic acid of a1) or b1) above due to the degeneracy of the genetic code;
Includes a nucleic acid selected from the group consisting of wild type, a microorganism whose genome is present in the same state as before the introduction of modifications ptsA gene fingers,
The modified microorganism as described above, wherein the modified microorganism belongs to the genus Basphia . The microorganism is genetically modified in the following A) to D):
A) Deletion of the ldhA gene or at least a part thereof, deletion of a regulatory element of the ldhA gene or at least a part thereof, or introduction of at least one mutation into the ldhA gene;
B) a deletion of the pflD gene or at least part thereof, a deletion of a regulatory element of the pflD gene or at least part thereof, or introduction of at least one mutation into the pflD gene; or
deletion of the pflA gene or at least part thereof, deletion of regulatory elements of the pflA gene or at least part thereof, or introduction of at least one mutation into the pflA gene;
C) Deletion of the wcaJ gene or at least part thereof, deletion of a regulatory element of the wcaJ gene or at least part thereof, or introduction of at least one mutation into the wcaJ gene;
D) at least one deletion of the pykA gene or at least a part thereof, a deletion of a regulatory element of the pykA gene or at least a part thereof, or at least one mutation to the pykA gene, which at least reduces the activity of the enzyme encoded by the pykA gene Introduction of;
The modified microorganism according to claim 1, comprising at least one of the following. The microorganism is:
A) Deletion of the ldhA gene or at least part thereof;
B) Deletion of pflA gene or at least part thereof, or deletion of pflD gene or at least part thereof;
C) introduction of at least one mutation into the wcaJ gene that results in the expression of the enzyme encoded by the truncated wcaJ gene;
D) introduction of at least one mutation into the pykA gene; and
E) Deletion of the ptsA gene or at least part thereof;
The modified microorganism according to claim 2 , comprising: I) cultivating the modified microorganism according to any one of claims 1 to 3 in a culture medium comprising at least one assimilable carbon source allowing the modified microorganism to produce organic compounds, thereby Obtaining a fermentation broth comprising an organic compound;
II) A method for producing an organic compound comprising the step of recovering the organic compound from the fermentation broth obtained in process step I). I) A modified microorganism of the genus Basfia having reduced activity of the enzyme encoded by the ptsH gene compared to the wild type, wherein the reduction of the activity of the enzyme encoded by the ptsH gene is achieved by modification of the ptsH gene The modified microorganism, wherein the wild type refers to a microorganism whose genome exists in the same state as before the introduction of the modification of the ptsH gene,
Culturing in a culture medium comprising at least one assimilable carbon source that enables the modified microorganism to produce organic compounds, thereby obtaining a fermentation broth comprising the organic compounds;
Comprising the step of recovering the organic compound from the fermentation broth obtained in II) Process step I), an organic compound Ri der succinic acid,
Based on the total weight of the assimilable carbon source excluding carbon dioxide, at least 50% by weight of the assimilable carbon source is sucrose;
Production method of organic compounds. The method according to claim 4 , wherein the organic compound is succinic acid. The method comprises process steps:
III) Conversion of the organic compound contained in the fermentation broth obtained in process step I) to a secondary organic product different from the organic compound by at least one chemical reaction, or recovered organic obtained in process step II) The method according to any one of claims 4 to 6 , further comprising conversion of the compound. The organic compound is succinic acid and the secondary organic product is selected from the group consisting of succinic acid esters or polymers thereof, tetrahydrofuran (THF), 1,4-butanediol (BDO), γ-butyrolactone (GBL) and pyrrolidone The method according to claim 7 . Use of the modified microorganism according to any one of claims 1 to 3 for fermentative production of an organic compound. Use according to claim 9 , wherein the organic compound is succinic acid and sucrose is used as an assimilable carbon source.